The long term goal of this continuing project is to learn how the biosynthesis of pyridoxine (vitamin B6;PN) and pyridoxal phosphate (PLP) are regulated at the pathway and genetic levels and integrated into general cellular metabolism. Pyridoxal phosphate is an essential, ubiquitous coenzyme that plays numerous roles in cellular metabolism, particularly of amino acids, in all organisms. Considerable evidence suggests that PN and PLP concentrations are strictly regulated at the pathway and genetic levels; yet, relatively little has been proven about the mechanisms that bring about this regulation. In this regard, Escherichia coli is an ideal model organism for these broadly based physiological, genetic, and biochemical studies, because it synthesizes PN, like plants and certain other microorganisms, and then converts PN into PLP by a two-step pathway that seems to be universal. Four Specific Aims are planned for this five-year proposal. First, investigation will be continued of the regulation of PN biosynthesis. Aim I includes analysis of PdxB and SerA molecular evolution, biochemical characterization of Pdx enzyme functions, continued molecular genetic analyses of the structure and regulation of interesting pdx complex operons, development of promising genetic approaches to isolate pdx regulatory mutants, and genetic and biochemical examination of a pathway that may provide a shared precursor for vitamins B1, B2, and B6 biosynthesis. Second, the regulation of the universal pathway from PN to PLP will be studied further. Aim II includes identification, mapping, isolation, and regulatory analyses of the essential pdxK (PN/PL kinase) gene and the pdxT (PN/PL facilitator), and pdxD (PL dehydrogenase) genes, critical evaluation of several hypotheses about the regulation of the PN PNP PLP pathway, and genetic and enzymological characterization of pdxH (PNP oxidase). Third, B6 vitamer and PLP levels will be measured in pdx mutants and in bacteria grown under a variety of stress conditions, including ones that cause large inductions of PLP-requiring enzymes. Fourth, the PN/PL facilitator will be characterized to learn whether E. coli uses a pore or carrier for diffusion of ringed compounds. The continuation of this project is important for several reasons. It provides basic knowledge about the regulation of coenzyme biosynthesis in a physiologically and genetically tractable organism. Current experiments are at the point of critically testing several key hypotheses about how PN and PLP biosynthesis is regulated. The project is providing significant new information about mechanisms that integrate coenzyme biosynthesis into general cellular metabolism. Several aspects of the project transcend coenzyme biosynthesis and are offering insights into topics of fundamental biological interest, such as the structure and regulation of complex operons and the evolution of biosynthetic pathways. Finally, increased knowledge about PLP biosynthesis is of obvious biomedical and biotechnological relevance, because PLP plays well-documented, diverse roles in human intermediary metabolism, physiology, and disease.